This application proposes the continuation of our investigation of the molecular and biological functions of the vitamin D hormone (1,25(OH)2D3) in cultured cell systems. There will be a new focus on 1,25(OH)2D3 action in osteoblast-like bone cells and epithelial cells of the intestine, where vitamin D exerts its major effects upon bone remodeling and mineral absorption, respectively. In the last grant period we accomplished the molecular cloning, overexpression and immunocytochemical localization of the human vitamin D receptor (hVDR), and elucidated the vitamin D response element (VDRE) in the rat osteocalcin gene. These findings will be extended to probe the phosphorylation of hVDR in relation to its ability to bind to the VDRE and enhance transcription of an osteocalcin promoter/reporter construct in transfected cells. Site-directed mutagenesis will be employed to determine the residues in hVDR that are phosphorylated and stimulation of the relevant protein kinases in intact cells will be effected to test in vivo significance. Next, hVDR will be overexpressed in the insect baculovirus system and in E. coli to generate sufficient quantities for biochemical study. Expressed hVDR will be purified for study of phosphorylation by protein kinase C and casein kinase II, in vitro. Phosphopeptide mapping of hVDR overexpressed in COS-7 cells will be used to elucidate the site of 1,25(OH)2D3-dependent, hVDR phosphorylation. Preliminary data that a nuclear receptor auxiliary factor (RAF) is required for high-affinity VDR binding to the VDRE will be pursued by characterizing, purifying and cloning the major RAF in CV-1 cells. RAF will be cloned by screening an expression vector library with purified hVDR/[32p]VDRE. Ultimately, the above components will be reassembled as purified factors in an in vitro transcription system designed to determine the mechanism whereby 1,25(OH)2D3 stimulates the tissue specific expression of osteocalcin. Finally, a molecular comparison of 1,25(OH)2D3/VDR action on osteocalcin will be made with the effect on calbindin-D28k (CaBP) induction in cultured embryonic duodena. Because it is inhibited by cycloheximide, CaBP regulation by 1,25(OH)2D3 may be unique in that an intermediary induced protein may bind to a non-VDRE enhancer in the CaBP promoter and/or stabilization of CaBP mRNA may occur. In toto, these experiments should define the precise signal transduction pathways whereby vitamin D controls the expression of crucial 'bone and intestinal genes, and these results may provide clues to the molecular basis of clinical vitamin D resistance as well as to the role of vitamin D in the prevention of osteoporosis.